Metastasis is responsible for most cancer-related mortality although it remains a poorly understood process. Metastatic tumor cells extravasate from the primary tumor and colonize in distant tissues with little understanding about mechanisms regulating colonization. The role of the vasculature in pre-metastatic sites and metastatic progression has not been extensively examined. Recent work from our lab shows that the calcineurin-NFAT pathway is activated specifically in lung endothelium prior to the detection of tumor cells that preferentially metastasize to the lung. Our preliminary data implicates endothelial activation as a prerequisite for metastatic colonization with this effect mediated at least partially via activation of the calcineurin-NFAT signaling pathway. Calcineurin, a ser/thr phosphatase, activates NFAT family transcription factors to regulate the development of many tissues. We and others have shown that the calcineurin-NFAT pathway is a key intracellular mediator of VEGF-A signaling in endothelial cells and angiogenesis. The importance of calcineurin signaling in tumor angiogenesis is illustrated by the remarkable protection against solid tumors observed in Down syndrome (DS) individuals. Our previously published studies in a Down syndrome mouse model indicate that defects in tumor angiogenesis resulting in significantly decreased tumor growth is due in part to attenuation of calcineurin signaling in endothelial cell by the increased expression of chromosome 21-encoded inhibitors of this pathway, Down syndrome candidate region-1 (Dscr1) and Dual specificity kinase 1A (Dyrk1A). Here we propose to explore novel clinically relevant roles for this signaling pathway and two specific downstream targets, thrombospondin-1 (TSP-1) and angiopoietin-2 (ANG-2) in activating the vascular endothelium in pre-metastatic sites in the lung and liver. We hypothesize that endothelial activation primes pre-metastatic sites providing a receptive microenvironment for the engraftment and survival of metastatic tumor cells. We will genetically and pharmacologically manipulate the calcineurin-NFAT pathway, TSP-1 and ANG-2 expression in endothelial cells to determine the effects on lung and liver metastasis. We will utilize transgenic mouse models of primary tumors that spontaneously metastasize to the lung or liver as well as lung and liver colonization assays to investigate metastatic tumor cell engraftment. Our studies will better elucidate the biology of calcineurin-NFAT-TSP-1 and ANG-2 signaling in endothelial cells at metastatic sites and mechanisms underlying metastatic tumor cell colonization in distant tissues. These findings may also lead to new therapies to prevent widespread lung and liver metastasis.